To date, in vivo molecular imaging agents specifically target the cell surface or microenvironment. However, most of the highly specific changes of cancer cells that differentiate cancers cells from normal cells occur intracellularly. The challenge, therefore, is to develop agents that report intracytoplasmic changes yet still are capable of being imaged in vivo. The first step in achieving this goal is to target the imaging agent to the cell surface which requires affinity for a cell surface marker. The ligand must then be internalized by endocytosis and then bind to the appropriate site whereupon it activates. These requirements place large demands on synthetic chemistry since the molecular construct must have multiple functionalities. We are developing smart activatable optical constructs which only fluoresce when they are internalized to the cytoplasm. Using a series of commercially available dyes that are bound to targeting compounds and then modified to fluoresce under specific intracellular conditions such as lower pH and in the presence of specific enzymatic activity we are making progress toward the goal of intracellular in vivo imaging. This work is being performed in collaboration with Prof. Urano from the University of Tokyo Chemistry Department. Over the past year we have made considerable advances in this area by proving that it is possible to create highly activatable optical imaging agents based on the BODIPY and Rhodamine backbones. We are also pioneering efforts to create multimodal imaging agents;agents that can be seen on both optical cameras as well as PET, MR or radionuclide cameras. The agents being designed are highly biocompatible and elements have already been used in humans. For instance, the agent Galactosylserum Albumin (GSA) which we have labeled with Rhodamine Green (GSA-RhG) is internalized rapidly within cancer cells and may be viable as an agent for human use. We are developing activated fluorescent molecular imaging agents and have a number of successes over the year. However, we continue to pursue a solution that will activate only within cancer cells and not within other, normal cells. In addition to GSA as a targeting ligand we are employing commercially available antibodies such as trastuzumab and cetuximab that will enable targeting, binding and internalization. Specifically, we have used trastuzumab in combination with a self quenched indocyanine green, ICG, an FDA approved Near InfraRed (NIR) dye to target in situ tumors. When the antibody binds its cognate receptor, it is internalized releasing the ICG which then begins to fluoresce. Both components of this construct are FDA-approved so in theory this technique could be translated clinically fairly easily. The ability to image multiple targets simulanteously led us to explore multiexcitation and multiemission cameras. We had hoped that a single excitation light would be able to activate multiple fluorophores at differing wavelengths but this proved to be unrealistic. Instead, we use multiple wavelength excitation light using a new Maestro camera. This has allowed us to simultaneously image up to 4 targets in the near infrared and is very promising for clinical application. Additionally, we are developing fiberoptic scopes with fluorescence receptors to allow very small areas to be examined percutaneously using small fiber-based scopes. Recently, we have demonstrated that is possible to image live unanesthetized mice by using a highly tuned real time camera. It may be possible in the near future to develop multi-targeted multi color imaging to better characterize tumors. Recently, we have used the Halotag technology to perform in vivo imaging. Currently, if researchers want to label cells with a fluorescent marker (such as Green Fluorescent Protein (GFP)), they must transfect the cell with a suitable gene. If their needs for color imaging change (i.e., they need to label the cells red so as not to compete with a drug that fluoresces in the green) they must rederive a cell line that expresses Red Fluorescent Protein (RFP). Halotag is a genetic construct that leads to a special, non-natural enzyme expression on the cell surface. A Halo ligand can then bind this enzyme avidly. By attaching different color fluorophores to each Halo ligand it is possible to change the colors of the cells without rederiving the cell line, a potential advantage in multicolor experiments. Moreover, recent results in our lab demonstrate that is possible to get excellent imaging results by simply exchanging the targeting Halo ligand with another fluorophore. This method should add flexibility to similar experiments. Most recently, we have developed highly specific activatable probes based on H dimer formation. For instance, Rhodamine dimerizes when bound to the Fc portion of an antibody leading to a quenching of fluorescence. pH activatable probes have been developed as well as upconverting nanocrystals that are excited at higher wavelengths than they emit at. Finally, uniquely targeted near infrared probes have been developed that appear to have specific cell membrane toxicity suitable for photodynamic therapy that is highly targeted.